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Dissolution of lysozyme-coated hydroxyapatite
COLLOIDS AND SURFACES ELSEVIER
B
Colloids and Surfaces B: Biointerfaces 7 (199611 8
Dissolution of lysozyme-coated hydroxyapatite F. Poumier a, Ph. Schaad a, y. Haikel a J.C. Voegel a, Ph. Gramain b,, Centre de Recherches Odontologiques, I.N.S.E.R.M. U. 424, 1 place de I'H6pital, 67000 Str~lsbourg, Fr~nce b E~ole Nationale Sup~rieure de Chimie, Laboratoire de Chimie Appliqu~e, CNRS-URA 11 93. ~ rue de l'Ecole Normclle, 34053 Montpellier Cedex I, Franc~'
Received 19 June 1995; accepted 11 March 1996
Abstract The effect of the adsorption of chicken egg-white lysozyme, a basic protein, on the dissolution kinetics of synthetic hydroxyapatite (HAP) is studied at a constant pH of 5.0. It is confirmed that lysozyme adsorption occurs in spite of the positive net charge of the HAP surface and is favoured by high concentrations of phosphate ions. This implies that adsorption sites for the protein are phosphate ions adsorbed onto HAP. The dissolution kinetics performed with lysozyme-coated HAP in the presence of the protein in solution show no significant difference from kinetics obtained with uncoated HAP surfaces. This absence of effect is explained by considering the limited coverage of the HAP by the protein, whose low molecular weight, globular shape, rigidity and stability lead to a small number of anchoring points. Keywords: Dissolution: Hydroxyapatite; Lysozyme
I. Introduction The presence of the acquired enamel pellicle layer, consisting mainly of proteins, contributes to tooth protection [-1-3]. Elucidating the roles of proteins in the mineralization/demineralization process requires the understanding of proteinhydroxyapatite interactions. This necessitates the characterization of protein adsorption together with the study of the effect of this protein on the H A P dissolution process. As of yet, studies concerning the role of salivary proteins in H A P demineralization are scarce [ 4 - 6 ] but it has been demonstrated that both acidic and basic proteins interact significantly with the H A P surface [7-11 ]. Further, it has been shown that acidic proteins such as albumin [4,5] may act as active inhibitors * Corresponding author. 0927-7765/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PI1 S0927-7765(96)01277-5
of demineralization. However, to our knowledge, the effect of the adsorption of basic proteins on H A P dissolution has not been studied. The aim of the present study was to investigate the influence on H A P dissolution of a basic protein, chicken egg-white lysozyme. Lysozyme is a small, rigid enzyme known for its antibacterial properties and commonly found in saliva. The adsorption of lysozyme onto HAP, previously well-described [ 11], will be discussed briefly and its influence on the kinetics of H A P dissolution at a constant pH of 5.0, in solution containing (and not containing) the protein, will also be described. 2. Materials and methods 2.1. Hydroxyapatite powder
All the experiments were performed with 100-165~tm hydroxyapatite platelets (Bio-Gel
2
F. Poumier et al./Colloids Surfaces B: Biointerfaces 7 (1996) 1 8
HTP, Bio-Rad Laboratories, Richmond, CA) prepared as described previously by Tiselius et al. [12]. The specific surface area, determined by the BET method for HAP, was 51 __+11 m 2 g-1. This high value is the consequence of the rugosity of the platelet-shaped material. Calcium and phosphate analyses led to a Ca/P ratio for the HAP sample of 1.62 _+0.09, close to the theoretical value of 1.67. The solubility product (pKsp) at 37°C and in the presence of 8 × 10 -2 M KCI was found to be 59.5_+ 1.2 [13] and can be compared with published values 1-14] obtained at lower ionic strength and at 25°C, ranging from 55 to 60 with a most probable value of 57.5. The higher value obtained at 37°C is in agreement with the generally observed solubility decrease with increasing temperature.
Table 1 Composition of the different solutions used Solutions useda
pH +0.020f equilibrium
CaCI 2 KH2PO4 Ca/P (M x 103) (M x 103) ± 10% ___5% + 5%
Ab Bb
7.0 7,0 7,0 5.0 5.0 5.0
0.10 0,50 0.10 5.0 25.0 5.0
Cb
Dc E~ Fc
0.06 0.06 1.0 3.0 3.0 50.0
1.67 8.34 0.1 1.67 8.34 0.1
All solutions contain 8 x 10 2 M KCI. b Solution corresponded to HAP saturation at pH 7.0 at different Ca/P ratios. c Solution corresponded to HAP saturation at pH 5.0. "
Chicken egg-white lysozyme was supplied by Sigma (L 6876, grade I). It is a well-characterized small and rigid protein (Mw ~ 14500; dimensions 4.5 × 3 x 3 nm3), very stable in solution [153 with a high isoelectric point (pHi = 11.0-11.3).
required composition. Then, after HAP settling, the volume of the solution was reduced to 10 ml, the required amount of labelled lysozyme added and adsorption pursued for 2 h, At the end of this stage, a fraction of the supernatant was withdrawn (200 tal) and accurately weighed. Its activity was measured and the amount of adsorbed protein determined. Each experiment was repeated at least twice.
2.3. Lysozyme adsorption experiments
2.4. HAP dissolution set-up
The quantity of lysozyme adsorbed onto the HAP was determined by measuring the depletion of radiolabelled protein solutions. The protein was labelled with 125I using the iodine monochloride method [16]. All solutions were prepared with deionized water. The concentration of the labelled protein solution was determined by absorbance at 280 nm and the specific activity (cpm mg -1) was measured by ~, counting (Packard, United Technologies). Adsorption experiments in the presence of an initial protein concentration of 1.5 x 10 -2 wt.% were performed at pH 5.0 and 7.0, at 22 + I°C, with different solutions (see Table 1) with Ca/P ratios equal to, higher or lower than the stoichiometric value of 1.67; for each ratio, the quantity of calcium and phosphate were chosen to correspond to HAP saturation. Thus, 10 mg of HAP were preequilibrated for 2 h in an argon atmosphere (without proteins) within 50ml of solution at the
Dissolution experiments at constant pH were performed in an argon atmosphere with automatic equipment described previously [17,18]. The reliability and reproducibility of the apparatus, the behaviour of the electrodes, the correction for slow evaporation (0.6 vol.% after 6 h) the acid injection, the data acquisition and the data processing system were all discussed in detail. Briefly, the equipment included (i) a closed double-walled 100 ml capacity Pyrex glass reactor thermostatted at 37°C (Mettler, Greifensee, Switzerland) equiped with a well-controlled stirring system; (ii) a combined pH electrode (Mettler, type DG-111, Greifensee, Switzerland) connected to a DL-21 Mettler pH-stat unit, which continuously follows the H + consumption; (iii) a calcium electrode connected to another pH-stat unit which continuously follows the variation of calcium activity; (iv) a dual-channel recorder and a microcomputer. The experimental error in the determination of
2.2. Lysozyme
F. Poumier et aL/Colloids Surfaces B: Biointerlaces 7 (1996) 1 8
the calcium concentration was about 4%, with an error of 1.5% on consumed protons. The reproducibility of the dissolution experiments is of the order of 10%.
2.5. Determination of the point of zero charge ( PZC ~ ofhydroxyapatite The PZC of the electrical double layer of free charges for the hydroxyapatite interface was determined from the H + and O H - adsorption isotherms, obtained at 37°C from acid and base potentiometric titration curves [ 19]. Acid and base titrations were conducted in the dissolution set-up described above. After the addition to the reactor of 50 ml of standard solution A (Table 1), which contained 8 x 1 0 - 2 M KC1 and stoichiometric amounts of calcium and phosphate corresponding to HAP saturation at pH 7.0, the electrodes were stabilized for 1 h and 20 mg of HAP were introduced into the solution. After a 2 h equilibration period, the composition of the solution was noted (pH 7.2 _ 0.2; 1.42 x 10 4 ___5.7 +_ 10 - 6 M calcium and 8.75× 10 s + 3 . 5 × 10 -6 M phosphate) a n d a series of titrations was begun. The pH increase observed during the equilibration period resulted from the amphoteric behaviour of HAP powder [13]. Small 10btl increments of HC1 or K O H solutions at a concentration of 10- ~ M were added. The addition of acid or alkali never exceeded 0.3 ml for the investigated pH range. The ionic strength of the suspension thus remained globally constant during the titration procedure. The speed of titration was about 0.6 pH units per hour and the pH range covered was between pH 8.15 and 6.4. The amount of adsorbed H + (or O H ) ions was determined from the total consumed H + (or OH ) by withdrawing (or adding) the protons consumed (or released) by HAP dissolution (or re-precipitation). This contribution was estimated from released calcium, assuming that phosphate and calcium ions involved in dissolution (or re-precipitation) varied in a stoichiometric manner. Corrections were made for the variations with the pH of the different ionic phosphate forms.
2.6. Solutions All the salts used to prepare the different solutions were of analytical grade. The ionic products
3
for HAP were calculated from activity coefficients given by the Debye-Hiickel equation [20,21]. Of the complexes which can be present in the solutions, only CaHPO4 (pK=0.711 and K H P O 2 ( p K = 1.04) can reach significant concentrations. The presence of these complexes, which is not detected by the calcium electrode, was nevertheless neglected in the calculation of the HAP solubility product since the concentration of the complex is always less than 5% of the total phosphate concentration. The phosphate concentration was determined with an experimental error of 10% by the spectrophotometric analysis of molybdenum blue phosphate complexes [22] using a Beckman Model 34 spectrophotometer.
2.7. Dissolution ofl lysozyme-coated HAP 40 mg of HAP were introduced into the reactor containing (200 - x) ml of a solution prepared with the required amounts of calcium and phosphate ions corresponding to the HAP thermodynamic equilibrium conditions (Table 1 ). After 2 h stabilization while stirring, x ml of lysozyme solution at the required concentration was added so that the concentration in the bulk solution was 1.5x 1 0 2 w t , % or 5,0× 10 2wt.%. Since the addition of the lysozyme induced a weak pH increase of the order of 0.02 pH unit, the pH was readjusted. Adsorption was pursued for 2 h, more than enough time to attain equilibrium, as demonstrated by preliminary experiments using labelled lysozyme. The stirring was stopped in order to allow the powder to settle and 150ml of the supernatant solution was withdrawn to analyze the phosphate concentration. The 50 ml of solution remaining in the reactor, containing equilibrated HAP coated with lysozyme, was used for the dissolution experiments. These experiments were carried out either in the presence of or in the absence of protein in the dissolving solution. For the first method, the dissolution process was started by the rapid addition of the required amount of HC1 in order to attain pH 5.0 within a few seconds. During the whole dissolution experiment, the pH was maintained constant (+_0.02pH units) by acid injection: proton consumption as well as calcium release was
4
F. Poumier et al./Colloids Surfaces B: Biointerfaces 7 (1996) 1 8
continuously followed with time. In the second method, after HAP settling, all the supernatant was withdrawn and replaced by 50 ml of a lysozyme-free solution having the same ionic composition and adjusted to pH 7.0. After a few minutes of stabilization, the dissolution was started as described previously for the first method.
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5 3. Results and discussion
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Since the main factors affecting the dissolution process of HAP are the composition of the dissolving solution and the HAP surface state, two types of experiments have been performed to study the influence of lysozyme. First, we determined the PZC of the hydroxyapatite used and established the adsorption isotherms of the protein on HAP in experimental conditions similar to those of the dissolution experiments. Secondly, we carried out dissolution kinetics of lysozyme-coated HAP at a constant pH of 5.0. Two types of dissolution experiments were performed in order to characterize both the influence of the lysozyme adsorbed to the surface and that of the lysozyme present in the solutions. 3.1. The P Z C of H A P
When studying the adsorption of proteins, the knowledge of the PZC of the surface of HAP is essential since the PZC values of HAP reported by various authors show significant differences, which can be explained by the difference in both sample origins and experimental conditions. The H + and O H - adsorption curves for hydroxyapatite in 8 x 10 -2 M KC1, obtained by titration after 2 h equilibration, are shown in Fig. 1. The intersection of the curves indicates that the pH at the PZC is 7.47. This value is in good agreement with the results of Somasundaran et al. [19] (PZC of 7.0) and of Barroug et al. [11] (PZC of 6.9 and 7.2) using an electrophoretic method. However, with an HAP sample of the same type as in this study, also supplied by Bio-Rad Laboratories, the latter authors found a value of 5.5. Two reasons may explain such a difference. First, as suggested by Barroug et al. [11], their
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Fig. 1. Effect of pH on the amounts of H ÷ and O H - adsorbed onto 20 mg of H A P equilibrated in a 8 x 10 2 M KC1 solution.
HAP sample, unlike our own, was calcium deficient with a Ca/P ratio of 1.5, which may have an important influence on the state surface. Secondly, before we used our Bio-Rad sample, it was first sieved several times, washed with methanol and dried at 40°C, to clean the HAP surface. 3.2. Lysozyme adsorption
Adsorption of lysozyme on apatite has been studied in detail by Barroug et al. [ 11 ]. Therefore, our main objective was to characterize the adsorption in experimental conditions close to those of our dissolution experiments carried out in the presence of the protein. Adsorption experiments of lysozyme on HAP were realized at pH 5.0 and 7.0 using an initial protein concentration of 1.5 x 10 -2 wt.% and solutions containing different Ca/P ratios (see Table 1). HAP was first preequilibrated in the solutions, then the protein was introduced. Its concentration in solution was determined after 2 h equilibration under slow agitation. This duration was chosen since preliminary experiments indicated that the saturation of adsorption of lysozyme on H A P was reached after less than 1 h of reaction. The results are shown in Fig. 2 together with the results of Barroug et al. [11] obtained at pH 6.8. In spite of the positive net charge of the HAP surface in the pH domain 5.0-7.0, the positively charged lysozyme is adsorbed in appreciable amounts, confirming the
E Poumier et al.,,'Colloids Surfaces B. Biointerfaces 7 (1996) 1 8 0.12
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previous results. The maximum adsorbed amounts increase with decreasing the Ca/P ratio. Since Barroug et al. [ 11 ] have shown that the maximum amounts and the affinity constant rise as phosphate concentration increases, the variation observed in Fig. 1 is interpreted as due to an increase of the phosphate concentration in the solution and the competition for adsorption sites of lysozyme with calcium ions. The higher adsorbed amounts obtained at pH 5.0 and at lower Ca/P ratios are well explained by considering the higher phosphate concentrations present in these conditions. The direct relationship between phosphate ions and lysozyme in the adsorption process indicates that phosphate ions are adsorbed onto the HAP surface and constitute the adsorption sites for the basic functional groups of lysozyme in competition with cations of the solution, as earlier suggested by Van Dulm et al. [23]. This is confirmed by the observation that the adsorption of each lysozyme molecule is accompanied by adsorption of about one phosphate ion [ 11 ]. Moreover, our results, which also show that no further lysozyme adsorption occurs at pH 5.0 when the phosphate concentration is increased from 3.0 × 1 0 - 3 M to 5.0 × 10 2 M (Fig. 2), suggest that the HAP surface becomes saturated with phosphate ions in this concentration
5
range. The data at pH 7.0, however, clearly demonstrates that adsorption of lysozyme is linked to the adsorption of phosphate ions. This conclusion is of prime importance not only for understanding the variations of the different adsorption parameters such as adsorbed amounts, affinity constant and reversibility but also for interpreting the results of the dissolution experiments described in the following section. At this point, it is interesting to consider the work of Nagadome et al. [24]. Using hydrogen exchange and l H NMR, these authors have shown that the contact surface area between the lysozyme adsorbed and the HAP surface is small, with only three to four residues of the enzyme bound to the crystal surface. Moreover, the NH protons involved are concentrated in the same part of the molecule and are located at the back surface of the active site, in agreement with the observation that the enzymatic activity for the lysozyme bound to HAP is not suppressed [25]. According to this result and considering that this enzyme possesses a high structural stability and a strong internal coherence [26], the adsorbed lysozyme molecule may be represented as an egg laid down on a flat surface. Using the stereo-view of lysozyme for an estimate [24], it was calculated that the surface area in contact with the HAP surface represents about 1.8 nm 2 per molecule. This represents only 1./5 of the total surface area occupied by an adsorbed enzyme. Considering the maximum amount of lysozyme adsorbed, we see that only 9% of the apatite surface is in close contact with the enzyme through phosphate ions bridges. 3.3. Dissolution kinetics oflysozyme-coated H A P
As already mentioned, two types of dissolution experiments were carried out at a constant pH of 5.0 from HAP slurries equilibrated at pH 7.0 in the presence of lysozyme at two concentrations. In the first type of experiment, the lysozyme in equilibrium with the HAP-coated powder was kept in the dissolving solution. In the second type, the protein was replaced after careful settling by a solution corresponding to the composition of solution A (Table 1 i. In both cases, the pH of 5.0 was attained within a few seconds by a rapid addition of HC1.
6
F Poumier et al./Colloids' Surfaces B." Biointerfaces 7 (1996) 1-8 5.0
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Fig. 3. Proton consumption (a) and calcium release (b) versus time for HAP dissolution performed at pH 5.0, 37°C, 1000 rev min-1 with 40 mg HAP pre-equilibrated in solution A, in the presence of lysozyme at concentrations of (1) 0, (2) 1.5 x 10 2 and (3) 5.0 x 10 -2 wt.%. Dissolutions were performed with the lysozyme in equilibrium in the dissolving solutions. Horizontal lines represent a degree of reaction of 30%.
Fig. 3 represents the kinetics of the calcium released and protons consumed in the case where lysozyme is present both at the HAP interface and in the dissolving solutions. Fig. 4 represents the same experiments but without protein in solutions. For each kinetics curve, the limit of 30% of reaction is indicated since up to this degree of conversion the variation of HAP surface area can be neglected
[27-]. In order to check for any influence of the protein on the composition of the dissolving solutions due to interactions with ions, total calcium and phosphate concentrations in solution were determined 10 h after the start of the dissolution kinetics, to
Time (rain.)
Fig. 4. Proton consumption (a) and calcium release (b) versus time for HAP dissolution performed at pH 5.0, 37°C, 1000 rev min-~ with 40 mg HAP pre-equilibrated in solution A, in the presence of lysozyme at concentrations of (1) 0, (2) 1.5 x 10 -2 and (3) 5.0 x 10 -2 wt.%. Dissolutions were performed after replacement of the equilibration solutions containing the protein by 50 ml of a protein-free solution A. Horizontal lines represent a degree of reaction of 30%.
be sure that HAP solubility equilibrium was attained. The results gathered in Table 2 show that, within the limits of the experimental errors, all the dissolutions can be considered as stoichiometric and that HAP saturation is reached. In other words, the lysozyme, in the range of concentrations used, does not interact sufficiently with the ions present in solutions to affect the normal HAP equilibrium. Since the most probable interactions concern lysozyme and phosphate ions, our results demonstrate that only weak electrostatic interactions occur, which is in agreement with the adsorption results. Coming back to the dissolution kinetics, one observes that all kinetics are comparable to the reference kinetics (curve 1 of Figs. 3 and
F Poumier et aL/Colloids Sur/ctces B." Biointe~ji, ces 7 " 1996) l~ ,~
Table 2 Concentrations of calcium and phosphate ions in the solutions at the equilibrium corresponding to the end of dissolution at pH 5.0 (after 10 hE. Dissolution experimentsperformed with (a) or without (b) lysozymeremaining in solution Initial protein concentration wt.~
[Ca2~] (Mxl0 a+4e4)
[P] pKsp (Mxl04+ 10%) (59.5+1.2)
0 1.5(a) 5.01al 1.5(b) 5.0tb)
35.5 34.5 32.6 32.3 35.3
22.0 24.0 22.5 22.6 22.9
59.7 59.6 59.8 59.9 59.7
4) whatever the protein concentration. In our experimental conditions, no effect on H A P dissolution is detected and even the presence of the protein at the interface during the dissolution process can be questioned. However, desorption of the protein would be surprising for three main reasons. First, the experiments shown in Fig. 3 are performed in the same conditions as our adsorption experiments, except for the pH jump from 7.0 to 5.0. This pH change may induce a change of adsorption behavior. However, we have shown that the amount of adsorbed lysozyme increases when pH decreases, which argues against a desorption process. Secondly, since adsorbed amounts increase with increasing phosphate concentration, the liberation of ions during the dissolution would favor the adsorption process during the acidic attack. Finally, a desorption mechanism induced by the stirring itself during the dissolution can be excluded considering the small size of H A P crystals and the well-known stagnant behavior of the Nernst layer adjacent to the H A P surface. Moreover, we have shown that, in the same experimental conditions, neither polymers [28,29] nor albumin [30] were desorbed, since they demonstrated inhibiting effects depending on the amounts adsorbed. We conclude that the lysozyme remains at the surface but that its presence in the dissolving solutions and/or at the HAP surface has no significant influence on the dissolution process. 4. Conclusion This study demonstrates that, although lysozyme is adsorbed onto the H A P surface, its presence has
7
no effect on the dissolution behavior of HAP, in contrast with various synthetic polymers and other proteins. It is interesting to remark that most of the efficient macromolecules are anionic, which suggests that the interactions between anionic groups and interfacial calcium ions are at the origin of the observed inhibition effect. This is clearly understandable since in the range of pH where inhibition occurs, the adsorbed substances and the apatite surface are of opposite charge. On this basis, the interaction of positive molecule with positive H A P can only take place through such negative ions as phosphate. This is illustrated with lysozyme in this study which demonstrates the determining role of interracial phosphate ions controlling the adsorption process. However, the purely electrostatic character of these interactions does not generate strong forces as with chelating groups. It is also clear that in conditions where both molecules and surface have the same net charge, obtaining a high surface coverage promoting the inhibition effect is difficult. We have seen that with lysozyme, the effective surface coverage is quite limited and that the number of contact points is also quite restricted. Such characteristics do not work ill favour of an inhibiting effect. In conclusion, on the basis of the experimental results and of the above discussion, the absence of effect of lysozyme on the dissohttion process of HAP, in our experimental conditions, is understandable. The mode of adsorption of the protein, its positive charge, its low molecular weight with few sites of contact with the HAP surface due to its rigidity and stability and the low ratio of surface coverage work against an inhibiting effect.
Acknowledgment Ph. Schaad wishes to thank the Facult~ d'Odontologie de Strasbourg for financial support.
References [ 1] J.L. Jensen, M.S. Lamkin and F.G. Oppenheim, I Dent. Res., 71 i 1992), 1569.
8
F Poumier et al./Colloids Surfaces R" Biointerfaces 7 (1996) 1-8
[2] R.T. Zahradnik, D. Propas and E.C. Moreno, J. Dent. Res., 56 (1977), 1107. [3] R.J. Gibbons, I. Etherden and W. Peros, in S.E. Mergenhagen and B. Rosan (Eds.), Molecular Basis of Microbial Adhesion, American Society of Microbiology, Washington, DC, 1985, p. 77. [4] J. Christoffersen, M.R. Christoffersen, P. Ibsen and H. Ipsen, J. Cryst. Growth, 18 (1986), 1. [5] K.O.A. Chin, M. Johnson, E.J. Bergey, M.J. Levine and G.H. Nancollas, Colloids Surfaces A: Physicochem. Eng. Aspects, 78 (1993), 229. [6] E.C. Reynolds, J. Dent. Res., 66 (1987), 1120. [7] W. Norde, Adv. Colloid Interface Sci., 25 (1986), 267. [8] V. Hladly and H. Fiiredi-Milhofer, J. Colloid Interface Sci., 69 (1979), 460. [9] M. Johnson, J.W. Perich, E.C. Reynolds and G.H. Nancollas, J. Colloid Interface Sci., 160 (1993), 179. [10] J.C. Voegel, S. Behr, M.J. Mura, J.D. Aptel, A. Scmitt and E.F. Bres, Colloids Surfaces, 40 (1989), 307. [11] A. Barroug, J. Lemaitre and P.G. Rouxhet, Colloids Surfaces, 37 (1989), 339. [12] A. Tiselius, S. Hjerten and O. Levin, Arch. Biocbem. Biophys., 65 (1956), 132. [13] P. Gramain, J.C. Voegel, M. Gumpper and J.M. Thomann, J. Colloid Interface Sci., 118 (1987), 148. [14] For a list of values obtained by different authors see for example: A.N. Smith, A.M. Posner and J.P. Quirk, J. Colloid Interface Sci., 54 (1976), 76.
[15] J.G.E.M. Fraaije, W. Norde and J. Lyklema, Biophys. Chem., 40 (1991), 317. [16] A.S. MacFarlane, J. Clin. Invest., 42 (1963), 346. [17] P. Gramain, J.M. Thomann, M. Gumpper and J.C. Voegel, J. Colloid Interface Sci., 128 (1989), 370. [18] J.M. Thomann, P. Gasser, E.F. Bres, J.C. Voegel and Ph. Gramain, Comput. Methods Progr. Biomed., 31 (1990), 89. [19] P. Somasundaran, J. Colloid Interface Sci., 27 (1968), 659. [20] J. Kieland, J. Am. Chem. Soc., 59 (1937) 1675. [21] G.G. Manov, R.G. Bates, W.J. Hamor and S.F. Acree, J. Am. Chem. Soc., 65 (1943), 1765. [22] B.N. Ames, Methods Enzymol., 8 (1966), 115. [23] P. Van Dulm, W. Norde and J. Lyklema, J. Colloid Interface Sci., 82 (1981), 77. [24] H. Nagadome, K. Kawano and Y. Terada, FEBS Letters, 317 (1993), 128. [25] K.M. Schilling and W.H. Bowen, J. Dent. Res., 67 (1988), 2. [26] T. Arai and W. Norde, Colloids and Surfaces, 51 (1990), 1. [27] J.M. Thomann, J.C. Voegel and P. Grarnain, Colloids and Surfaces, 54 (1991), 145. [28] Ph. Schaad, J.M. Thomann, J.C. Voegel and Ph. Gramain, Colloids and Surfaces A: Physicochem. Eng. Aspects, 83 (1994), 285. [29] Ph. Schaad, J.M. Thomann, J.C. Voegel and Ph. Gramain, J. Colloid Interface Sci., 164 (1994), 291. [30] F. Gorce, Ph. Schaad, Y. Haikel, J.C. Voegel and P. Gramain, submitted for publication.
B
Colloids and Surfaces B: Biointerfaces 7 (199611 8
Dissolution of lysozyme-coated hydroxyapatite F. Poumier a, Ph. Schaad a, y. Haikel a J.C. Voegel a, Ph. Gramain b,, Centre de Recherches Odontologiques, I.N.S.E.R.M. U. 424, 1 place de I'H6pital, 67000 Str~lsbourg, Fr~nce b E~ole Nationale Sup~rieure de Chimie, Laboratoire de Chimie Appliqu~e, CNRS-URA 11 93. ~ rue de l'Ecole Normclle, 34053 Montpellier Cedex I, Franc~'
Received 19 June 1995; accepted 11 March 1996
Abstract The effect of the adsorption of chicken egg-white lysozyme, a basic protein, on the dissolution kinetics of synthetic hydroxyapatite (HAP) is studied at a constant pH of 5.0. It is confirmed that lysozyme adsorption occurs in spite of the positive net charge of the HAP surface and is favoured by high concentrations of phosphate ions. This implies that adsorption sites for the protein are phosphate ions adsorbed onto HAP. The dissolution kinetics performed with lysozyme-coated HAP in the presence of the protein in solution show no significant difference from kinetics obtained with uncoated HAP surfaces. This absence of effect is explained by considering the limited coverage of the HAP by the protein, whose low molecular weight, globular shape, rigidity and stability lead to a small number of anchoring points. Keywords: Dissolution: Hydroxyapatite; Lysozyme
I. Introduction The presence of the acquired enamel pellicle layer, consisting mainly of proteins, contributes to tooth protection [-1-3]. Elucidating the roles of proteins in the mineralization/demineralization process requires the understanding of proteinhydroxyapatite interactions. This necessitates the characterization of protein adsorption together with the study of the effect of this protein on the H A P dissolution process. As of yet, studies concerning the role of salivary proteins in H A P demineralization are scarce [ 4 - 6 ] but it has been demonstrated that both acidic and basic proteins interact significantly with the H A P surface [7-11 ]. Further, it has been shown that acidic proteins such as albumin [4,5] may act as active inhibitors * Corresponding author. 0927-7765/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PI1 S0927-7765(96)01277-5
of demineralization. However, to our knowledge, the effect of the adsorption of basic proteins on H A P dissolution has not been studied. The aim of the present study was to investigate the influence on H A P dissolution of a basic protein, chicken egg-white lysozyme. Lysozyme is a small, rigid enzyme known for its antibacterial properties and commonly found in saliva. The adsorption of lysozyme onto HAP, previously well-described [ 11], will be discussed briefly and its influence on the kinetics of H A P dissolution at a constant pH of 5.0, in solution containing (and not containing) the protein, will also be described. 2. Materials and methods 2.1. Hydroxyapatite powder
All the experiments were performed with 100-165~tm hydroxyapatite platelets (Bio-Gel
2
F. Poumier et al./Colloids Surfaces B: Biointerfaces 7 (1996) 1 8
HTP, Bio-Rad Laboratories, Richmond, CA) prepared as described previously by Tiselius et al. [12]. The specific surface area, determined by the BET method for HAP, was 51 __+11 m 2 g-1. This high value is the consequence of the rugosity of the platelet-shaped material. Calcium and phosphate analyses led to a Ca/P ratio for the HAP sample of 1.62 _+0.09, close to the theoretical value of 1.67. The solubility product (pKsp) at 37°C and in the presence of 8 × 10 -2 M KCI was found to be 59.5_+ 1.2 [13] and can be compared with published values 1-14] obtained at lower ionic strength and at 25°C, ranging from 55 to 60 with a most probable value of 57.5. The higher value obtained at 37°C is in agreement with the generally observed solubility decrease with increasing temperature.
Table 1 Composition of the different solutions used Solutions useda
pH +0.020f equilibrium
CaCI 2 KH2PO4 Ca/P (M x 103) (M x 103) ± 10% ___5% + 5%
Ab Bb
7.0 7,0 7,0 5.0 5.0 5.0
0.10 0,50 0.10 5.0 25.0 5.0
Cb
Dc E~ Fc
0.06 0.06 1.0 3.0 3.0 50.0
1.67 8.34 0.1 1.67 8.34 0.1
All solutions contain 8 x 10 2 M KCI. b Solution corresponded to HAP saturation at pH 7.0 at different Ca/P ratios. c Solution corresponded to HAP saturation at pH 5.0. "
Chicken egg-white lysozyme was supplied by Sigma (L 6876, grade I). It is a well-characterized small and rigid protein (Mw ~ 14500; dimensions 4.5 × 3 x 3 nm3), very stable in solution [153 with a high isoelectric point (pHi = 11.0-11.3).
required composition. Then, after HAP settling, the volume of the solution was reduced to 10 ml, the required amount of labelled lysozyme added and adsorption pursued for 2 h, At the end of this stage, a fraction of the supernatant was withdrawn (200 tal) and accurately weighed. Its activity was measured and the amount of adsorbed protein determined. Each experiment was repeated at least twice.
2.3. Lysozyme adsorption experiments
2.4. HAP dissolution set-up
The quantity of lysozyme adsorbed onto the HAP was determined by measuring the depletion of radiolabelled protein solutions. The protein was labelled with 125I using the iodine monochloride method [16]. All solutions were prepared with deionized water. The concentration of the labelled protein solution was determined by absorbance at 280 nm and the specific activity (cpm mg -1) was measured by ~, counting (Packard, United Technologies). Adsorption experiments in the presence of an initial protein concentration of 1.5 x 10 -2 wt.% were performed at pH 5.0 and 7.0, at 22 + I°C, with different solutions (see Table 1) with Ca/P ratios equal to, higher or lower than the stoichiometric value of 1.67; for each ratio, the quantity of calcium and phosphate were chosen to correspond to HAP saturation. Thus, 10 mg of HAP were preequilibrated for 2 h in an argon atmosphere (without proteins) within 50ml of solution at the
Dissolution experiments at constant pH were performed in an argon atmosphere with automatic equipment described previously [17,18]. The reliability and reproducibility of the apparatus, the behaviour of the electrodes, the correction for slow evaporation (0.6 vol.% after 6 h) the acid injection, the data acquisition and the data processing system were all discussed in detail. Briefly, the equipment included (i) a closed double-walled 100 ml capacity Pyrex glass reactor thermostatted at 37°C (Mettler, Greifensee, Switzerland) equiped with a well-controlled stirring system; (ii) a combined pH electrode (Mettler, type DG-111, Greifensee, Switzerland) connected to a DL-21 Mettler pH-stat unit, which continuously follows the H + consumption; (iii) a calcium electrode connected to another pH-stat unit which continuously follows the variation of calcium activity; (iv) a dual-channel recorder and a microcomputer. The experimental error in the determination of
2.2. Lysozyme
F. Poumier et aL/Colloids Surfaces B: Biointerlaces 7 (1996) 1 8
the calcium concentration was about 4%, with an error of 1.5% on consumed protons. The reproducibility of the dissolution experiments is of the order of 10%.
2.5. Determination of the point of zero charge ( PZC ~ ofhydroxyapatite The PZC of the electrical double layer of free charges for the hydroxyapatite interface was determined from the H + and O H - adsorption isotherms, obtained at 37°C from acid and base potentiometric titration curves [ 19]. Acid and base titrations were conducted in the dissolution set-up described above. After the addition to the reactor of 50 ml of standard solution A (Table 1), which contained 8 x 1 0 - 2 M KC1 and stoichiometric amounts of calcium and phosphate corresponding to HAP saturation at pH 7.0, the electrodes were stabilized for 1 h and 20 mg of HAP were introduced into the solution. After a 2 h equilibration period, the composition of the solution was noted (pH 7.2 _ 0.2; 1.42 x 10 4 ___5.7 +_ 10 - 6 M calcium and 8.75× 10 s + 3 . 5 × 10 -6 M phosphate) a n d a series of titrations was begun. The pH increase observed during the equilibration period resulted from the amphoteric behaviour of HAP powder [13]. Small 10btl increments of HC1 or K O H solutions at a concentration of 10- ~ M were added. The addition of acid or alkali never exceeded 0.3 ml for the investigated pH range. The ionic strength of the suspension thus remained globally constant during the titration procedure. The speed of titration was about 0.6 pH units per hour and the pH range covered was between pH 8.15 and 6.4. The amount of adsorbed H + (or O H ) ions was determined from the total consumed H + (or OH ) by withdrawing (or adding) the protons consumed (or released) by HAP dissolution (or re-precipitation). This contribution was estimated from released calcium, assuming that phosphate and calcium ions involved in dissolution (or re-precipitation) varied in a stoichiometric manner. Corrections were made for the variations with the pH of the different ionic phosphate forms.
2.6. Solutions All the salts used to prepare the different solutions were of analytical grade. The ionic products
3
for HAP were calculated from activity coefficients given by the Debye-Hiickel equation [20,21]. Of the complexes which can be present in the solutions, only CaHPO4 (pK=0.711 and K H P O 2 ( p K = 1.04) can reach significant concentrations. The presence of these complexes, which is not detected by the calcium electrode, was nevertheless neglected in the calculation of the HAP solubility product since the concentration of the complex is always less than 5% of the total phosphate concentration. The phosphate concentration was determined with an experimental error of 10% by the spectrophotometric analysis of molybdenum blue phosphate complexes [22] using a Beckman Model 34 spectrophotometer.
2.7. Dissolution ofl lysozyme-coated HAP 40 mg of HAP were introduced into the reactor containing (200 - x) ml of a solution prepared with the required amounts of calcium and phosphate ions corresponding to the HAP thermodynamic equilibrium conditions (Table 1 ). After 2 h stabilization while stirring, x ml of lysozyme solution at the required concentration was added so that the concentration in the bulk solution was 1.5x 1 0 2 w t , % or 5,0× 10 2wt.%. Since the addition of the lysozyme induced a weak pH increase of the order of 0.02 pH unit, the pH was readjusted. Adsorption was pursued for 2 h, more than enough time to attain equilibrium, as demonstrated by preliminary experiments using labelled lysozyme. The stirring was stopped in order to allow the powder to settle and 150ml of the supernatant solution was withdrawn to analyze the phosphate concentration. The 50 ml of solution remaining in the reactor, containing equilibrated HAP coated with lysozyme, was used for the dissolution experiments. These experiments were carried out either in the presence of or in the absence of protein in the dissolving solution. For the first method, the dissolution process was started by the rapid addition of the required amount of HC1 in order to attain pH 5.0 within a few seconds. During the whole dissolution experiment, the pH was maintained constant (+_0.02pH units) by acid injection: proton consumption as well as calcium release was
4
F. Poumier et al./Colloids Surfaces B: Biointerfaces 7 (1996) 1 8
continuously followed with time. In the second method, after HAP settling, all the supernatant was withdrawn and replaced by 50 ml of a lysozyme-free solution having the same ionic composition and adjusted to pH 7.0. After a few minutes of stabilization, the dissolution was started as described previously for the first method.
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Since the main factors affecting the dissolution process of HAP are the composition of the dissolving solution and the HAP surface state, two types of experiments have been performed to study the influence of lysozyme. First, we determined the PZC of the hydroxyapatite used and established the adsorption isotherms of the protein on HAP in experimental conditions similar to those of the dissolution experiments. Secondly, we carried out dissolution kinetics of lysozyme-coated HAP at a constant pH of 5.0. Two types of dissolution experiments were performed in order to characterize both the influence of the lysozyme adsorbed to the surface and that of the lysozyme present in the solutions. 3.1. The P Z C of H A P
When studying the adsorption of proteins, the knowledge of the PZC of the surface of HAP is essential since the PZC values of HAP reported by various authors show significant differences, which can be explained by the difference in both sample origins and experimental conditions. The H + and O H - adsorption curves for hydroxyapatite in 8 x 10 -2 M KC1, obtained by titration after 2 h equilibration, are shown in Fig. 1. The intersection of the curves indicates that the pH at the PZC is 7.47. This value is in good agreement with the results of Somasundaran et al. [19] (PZC of 7.0) and of Barroug et al. [11] (PZC of 6.9 and 7.2) using an electrophoretic method. However, with an HAP sample of the same type as in this study, also supplied by Bio-Rad Laboratories, the latter authors found a value of 5.5. Two reasons may explain such a difference. First, as suggested by Barroug et al. [11], their
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7
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HAP sample, unlike our own, was calcium deficient with a Ca/P ratio of 1.5, which may have an important influence on the state surface. Secondly, before we used our Bio-Rad sample, it was first sieved several times, washed with methanol and dried at 40°C, to clean the HAP surface. 3.2. Lysozyme adsorption
Adsorption of lysozyme on apatite has been studied in detail by Barroug et al. [ 11 ]. Therefore, our main objective was to characterize the adsorption in experimental conditions close to those of our dissolution experiments carried out in the presence of the protein. Adsorption experiments of lysozyme on HAP were realized at pH 5.0 and 7.0 using an initial protein concentration of 1.5 x 10 -2 wt.% and solutions containing different Ca/P ratios (see Table 1). HAP was first preequilibrated in the solutions, then the protein was introduced. Its concentration in solution was determined after 2 h equilibration under slow agitation. This duration was chosen since preliminary experiments indicated that the saturation of adsorption of lysozyme on H A P was reached after less than 1 h of reaction. The results are shown in Fig. 2 together with the results of Barroug et al. [11] obtained at pH 6.8. In spite of the positive net charge of the HAP surface in the pH domain 5.0-7.0, the positively charged lysozyme is adsorbed in appreciable amounts, confirming the
E Poumier et al.,,'Colloids Surfaces B. Biointerfaces 7 (1996) 1 8 0.12
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previous results. The maximum adsorbed amounts increase with decreasing the Ca/P ratio. Since Barroug et al. [ 11 ] have shown that the maximum amounts and the affinity constant rise as phosphate concentration increases, the variation observed in Fig. 1 is interpreted as due to an increase of the phosphate concentration in the solution and the competition for adsorption sites of lysozyme with calcium ions. The higher adsorbed amounts obtained at pH 5.0 and at lower Ca/P ratios are well explained by considering the higher phosphate concentrations present in these conditions. The direct relationship between phosphate ions and lysozyme in the adsorption process indicates that phosphate ions are adsorbed onto the HAP surface and constitute the adsorption sites for the basic functional groups of lysozyme in competition with cations of the solution, as earlier suggested by Van Dulm et al. [23]. This is confirmed by the observation that the adsorption of each lysozyme molecule is accompanied by adsorption of about one phosphate ion [ 11 ]. Moreover, our results, which also show that no further lysozyme adsorption occurs at pH 5.0 when the phosphate concentration is increased from 3.0 × 1 0 - 3 M to 5.0 × 10 2 M (Fig. 2), suggest that the HAP surface becomes saturated with phosphate ions in this concentration
5
range. The data at pH 7.0, however, clearly demonstrates that adsorption of lysozyme is linked to the adsorption of phosphate ions. This conclusion is of prime importance not only for understanding the variations of the different adsorption parameters such as adsorbed amounts, affinity constant and reversibility but also for interpreting the results of the dissolution experiments described in the following section. At this point, it is interesting to consider the work of Nagadome et al. [24]. Using hydrogen exchange and l H NMR, these authors have shown that the contact surface area between the lysozyme adsorbed and the HAP surface is small, with only three to four residues of the enzyme bound to the crystal surface. Moreover, the NH protons involved are concentrated in the same part of the molecule and are located at the back surface of the active site, in agreement with the observation that the enzymatic activity for the lysozyme bound to HAP is not suppressed [25]. According to this result and considering that this enzyme possesses a high structural stability and a strong internal coherence [26], the adsorbed lysozyme molecule may be represented as an egg laid down on a flat surface. Using the stereo-view of lysozyme for an estimate [24], it was calculated that the surface area in contact with the HAP surface represents about 1.8 nm 2 per molecule. This represents only 1./5 of the total surface area occupied by an adsorbed enzyme. Considering the maximum amount of lysozyme adsorbed, we see that only 9% of the apatite surface is in close contact with the enzyme through phosphate ions bridges. 3.3. Dissolution kinetics oflysozyme-coated H A P
As already mentioned, two types of dissolution experiments were carried out at a constant pH of 5.0 from HAP slurries equilibrated at pH 7.0 in the presence of lysozyme at two concentrations. In the first type of experiment, the lysozyme in equilibrium with the HAP-coated powder was kept in the dissolving solution. In the second type, the protein was replaced after careful settling by a solution corresponding to the composition of solution A (Table 1 i. In both cases, the pH of 5.0 was attained within a few seconds by a rapid addition of HC1.
6
F Poumier et al./Colloids' Surfaces B." Biointerfaces 7 (1996) 1-8 5.0
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Fig. 3. Proton consumption (a) and calcium release (b) versus time for HAP dissolution performed at pH 5.0, 37°C, 1000 rev min-1 with 40 mg HAP pre-equilibrated in solution A, in the presence of lysozyme at concentrations of (1) 0, (2) 1.5 x 10 2 and (3) 5.0 x 10 -2 wt.%. Dissolutions were performed with the lysozyme in equilibrium in the dissolving solutions. Horizontal lines represent a degree of reaction of 30%.
Fig. 3 represents the kinetics of the calcium released and protons consumed in the case where lysozyme is present both at the HAP interface and in the dissolving solutions. Fig. 4 represents the same experiments but without protein in solutions. For each kinetics curve, the limit of 30% of reaction is indicated since up to this degree of conversion the variation of HAP surface area can be neglected
[27-]. In order to check for any influence of the protein on the composition of the dissolving solutions due to interactions with ions, total calcium and phosphate concentrations in solution were determined 10 h after the start of the dissolution kinetics, to
Time (rain.)
Fig. 4. Proton consumption (a) and calcium release (b) versus time for HAP dissolution performed at pH 5.0, 37°C, 1000 rev min-~ with 40 mg HAP pre-equilibrated in solution A, in the presence of lysozyme at concentrations of (1) 0, (2) 1.5 x 10 -2 and (3) 5.0 x 10 -2 wt.%. Dissolutions were performed after replacement of the equilibration solutions containing the protein by 50 ml of a protein-free solution A. Horizontal lines represent a degree of reaction of 30%.
be sure that HAP solubility equilibrium was attained. The results gathered in Table 2 show that, within the limits of the experimental errors, all the dissolutions can be considered as stoichiometric and that HAP saturation is reached. In other words, the lysozyme, in the range of concentrations used, does not interact sufficiently with the ions present in solutions to affect the normal HAP equilibrium. Since the most probable interactions concern lysozyme and phosphate ions, our results demonstrate that only weak electrostatic interactions occur, which is in agreement with the adsorption results. Coming back to the dissolution kinetics, one observes that all kinetics are comparable to the reference kinetics (curve 1 of Figs. 3 and
F Poumier et aL/Colloids Sur/ctces B." Biointe~ji, ces 7 " 1996) l~ ,~
Table 2 Concentrations of calcium and phosphate ions in the solutions at the equilibrium corresponding to the end of dissolution at pH 5.0 (after 10 hE. Dissolution experimentsperformed with (a) or without (b) lysozymeremaining in solution Initial protein concentration wt.~
[Ca2~] (Mxl0 a+4e4)
[P] pKsp (Mxl04+ 10%) (59.5+1.2)
0 1.5(a) 5.01al 1.5(b) 5.0tb)
35.5 34.5 32.6 32.3 35.3
22.0 24.0 22.5 22.6 22.9
59.7 59.6 59.8 59.9 59.7
4) whatever the protein concentration. In our experimental conditions, no effect on H A P dissolution is detected and even the presence of the protein at the interface during the dissolution process can be questioned. However, desorption of the protein would be surprising for three main reasons. First, the experiments shown in Fig. 3 are performed in the same conditions as our adsorption experiments, except for the pH jump from 7.0 to 5.0. This pH change may induce a change of adsorption behavior. However, we have shown that the amount of adsorbed lysozyme increases when pH decreases, which argues against a desorption process. Secondly, since adsorbed amounts increase with increasing phosphate concentration, the liberation of ions during the dissolution would favor the adsorption process during the acidic attack. Finally, a desorption mechanism induced by the stirring itself during the dissolution can be excluded considering the small size of H A P crystals and the well-known stagnant behavior of the Nernst layer adjacent to the H A P surface. Moreover, we have shown that, in the same experimental conditions, neither polymers [28,29] nor albumin [30] were desorbed, since they demonstrated inhibiting effects depending on the amounts adsorbed. We conclude that the lysozyme remains at the surface but that its presence in the dissolving solutions and/or at the HAP surface has no significant influence on the dissolution process. 4. Conclusion This study demonstrates that, although lysozyme is adsorbed onto the H A P surface, its presence has
7
no effect on the dissolution behavior of HAP, in contrast with various synthetic polymers and other proteins. It is interesting to remark that most of the efficient macromolecules are anionic, which suggests that the interactions between anionic groups and interfacial calcium ions are at the origin of the observed inhibition effect. This is clearly understandable since in the range of pH where inhibition occurs, the adsorbed substances and the apatite surface are of opposite charge. On this basis, the interaction of positive molecule with positive H A P can only take place through such negative ions as phosphate. This is illustrated with lysozyme in this study which demonstrates the determining role of interracial phosphate ions controlling the adsorption process. However, the purely electrostatic character of these interactions does not generate strong forces as with chelating groups. It is also clear that in conditions where both molecules and surface have the same net charge, obtaining a high surface coverage promoting the inhibition effect is difficult. We have seen that with lysozyme, the effective surface coverage is quite limited and that the number of contact points is also quite restricted. Such characteristics do not work ill favour of an inhibiting effect. In conclusion, on the basis of the experimental results and of the above discussion, the absence of effect of lysozyme on the dissohttion process of HAP, in our experimental conditions, is understandable. The mode of adsorption of the protein, its positive charge, its low molecular weight with few sites of contact with the HAP surface due to its rigidity and stability and the low ratio of surface coverage work against an inhibiting effect.
Acknowledgment Ph. Schaad wishes to thank the Facult~ d'Odontologie de Strasbourg for financial support.
References [ 1] J.L. Jensen, M.S. Lamkin and F.G. Oppenheim, I Dent. Res., 71 i 1992), 1569.
8
F Poumier et al./Colloids Surfaces R" Biointerfaces 7 (1996) 1-8
[2] R.T. Zahradnik, D. Propas and E.C. Moreno, J. Dent. Res., 56 (1977), 1107. [3] R.J. Gibbons, I. Etherden and W. Peros, in S.E. Mergenhagen and B. Rosan (Eds.), Molecular Basis of Microbial Adhesion, American Society of Microbiology, Washington, DC, 1985, p. 77. [4] J. Christoffersen, M.R. Christoffersen, P. Ibsen and H. Ipsen, J. Cryst. Growth, 18 (1986), 1. [5] K.O.A. Chin, M. Johnson, E.J. Bergey, M.J. Levine and G.H. Nancollas, Colloids Surfaces A: Physicochem. Eng. Aspects, 78 (1993), 229. [6] E.C. Reynolds, J. Dent. Res., 66 (1987), 1120. [7] W. Norde, Adv. Colloid Interface Sci., 25 (1986), 267. [8] V. Hladly and H. Fiiredi-Milhofer, J. Colloid Interface Sci., 69 (1979), 460. [9] M. Johnson, J.W. Perich, E.C. Reynolds and G.H. Nancollas, J. Colloid Interface Sci., 160 (1993), 179. [10] J.C. Voegel, S. Behr, M.J. Mura, J.D. Aptel, A. Scmitt and E.F. Bres, Colloids Surfaces, 40 (1989), 307. [11] A. Barroug, J. Lemaitre and P.G. Rouxhet, Colloids Surfaces, 37 (1989), 339. [12] A. Tiselius, S. Hjerten and O. Levin, Arch. Biocbem. Biophys., 65 (1956), 132. [13] P. Gramain, J.C. Voegel, M. Gumpper and J.M. Thomann, J. Colloid Interface Sci., 118 (1987), 148. [14] For a list of values obtained by different authors see for example: A.N. Smith, A.M. Posner and J.P. Quirk, J. Colloid Interface Sci., 54 (1976), 76.
[15] J.G.E.M. Fraaije, W. Norde and J. Lyklema, Biophys. Chem., 40 (1991), 317. [16] A.S. MacFarlane, J. Clin. Invest., 42 (1963), 346. [17] P. Gramain, J.M. Thomann, M. Gumpper and J.C. Voegel, J. Colloid Interface Sci., 128 (1989), 370. [18] J.M. Thomann, P. Gasser, E.F. Bres, J.C. Voegel and Ph. Gramain, Comput. Methods Progr. Biomed., 31 (1990), 89. [19] P. Somasundaran, J. Colloid Interface Sci., 27 (1968), 659. [20] J. Kieland, J. Am. Chem. Soc., 59 (1937) 1675. [21] G.G. Manov, R.G. Bates, W.J. Hamor and S.F. Acree, J. Am. Chem. Soc., 65 (1943), 1765. [22] B.N. Ames, Methods Enzymol., 8 (1966), 115. [23] P. Van Dulm, W. Norde and J. Lyklema, J. Colloid Interface Sci., 82 (1981), 77. [24] H. Nagadome, K. Kawano and Y. Terada, FEBS Letters, 317 (1993), 128. [25] K.M. Schilling and W.H. Bowen, J. Dent. Res., 67 (1988), 2. [26] T. Arai and W. Norde, Colloids and Surfaces, 51 (1990), 1. [27] J.M. Thomann, J.C. Voegel and P. Grarnain, Colloids and Surfaces, 54 (1991), 145. [28] Ph. Schaad, J.M. Thomann, J.C. Voegel and Ph. Gramain, Colloids and Surfaces A: Physicochem. Eng. Aspects, 83 (1994), 285. [29] Ph. Schaad, J.M. Thomann, J.C. Voegel and Ph. Gramain, J. Colloid Interface Sci., 164 (1994), 291. [30] F. Gorce, Ph. Schaad, Y. Haikel, J.C. Voegel and P. Gramain, submitted for publication.